EP1363924A1

EP1363924A1 - Avilamycin derivatives

Info

Publication number: EP1363924A1
Application number: EP01969646A
Authority: EP
Inventors: Gabriele Weitnauer; Agnes MÜHLENWEG; Axel Trefzer; Andreas Bechthold
Original assignee: Combinature Biopharm AG
Current assignee: Combinature Biopharm AG
Priority date: 2001-02-25
Filing date: 2001-08-24
Publication date: 2003-11-26
Also published as: JP2004518803A; US20040006026A1; CA2439150A1; WO2002068436A1; DE10109166A1

Abstract

The invention relates to avilamycin derivatives, genetic engineering, biosynthetic methods for the production thereof, medicaments containing said compounds and the utilization of said compounds for the production of a medicament against infectious diseases. The invention also relates to the nucleic acids, proteins and gene clusters and corresponding cells connected to the production of said avilamycin derivatives.

Description

Avilamycin derivatives

The invention relates to avilamycin derivatives (hereinafter also referred to as gavibamycins), genetic engineering biosynthetic processes for their preparation, medicaments containing these compounds, and the use of these compounds for the production of a medicament, for example against infectious diseases, as well as nucleic acids, proteins and gene clusters and corresponding cells that are associated with the production of these avilamycin derivatives.

The emergence of pathogenic bacteria that are multi-resistant to antibiotics poses a growing threat to human health and has intensified the search for new active substances. Fewer and fewer new active ingredients have emerged in the course of target-specific active ingredient screening in the past two decades, so that researchers have started to use new technologies to produce new compounds in addition to the search for new antibiotic active ingredients. A promising new technology is called combinatorial Biosynthesis refers to and uses biosynthetic genes as a means of manufacturing new drugs.

Another class of compound that is interesting in this context is the orthosomycins. They are a well-known class of antibiotics that are produced by various Actinomycetes. Members of this class act on a wide range of gram-positive pathogenic bacteria, including glycopeptide-resistant enterococci, methicillin-resistant staphylococci and penicillin-resistant streptococci.

Prominent examples of orthosomycins are avilamycins and evemimycins, which are produced by Streptomyces viridochromogenes Tü57 and Micromonospora carbonacea, respectively. These antibiotics consist of a heptasaccharide side chain and a dichloroisoevernic acid derived from the polyketide (acetate units) as agiycon, the sugar residues being partly linked to one another via orthoester bonds. This bond gives the whole class of orthosomycins the name. The exact mechanism of action of Orthosomycine is unknown. While a cell membrane effect has been discussed for a particular orthosomycin (ziracin) (Walker, 1976; Langer, 1987), an interaction with the ribosomal protein L16 is mentioned in more recent publications (Foster and Rybak, 1999). For another orthosomycin, avilamycin A, an inhibition of protein biosynthesis is assumed and an inhibition of the translation-initiation complex is proposed (Wolf, 1973).

The well-known avilamycins were obtained in 1959 from culture filtrates from

Streptomyces viridochromogenes Tü57 isolated (Buzzetti, et al., 1968;

Mertz et al., 1986). As already indicated above, avilamycin A, one of the main components, is made up of sugars. Individual components are D- Olivose, 2-deoxy-D-evalose, 4-0-methyl-D-fucose, 2,6-di-O-methyl-D-mannose, L-lyxose and methyl eurekanate. In studies, avilamycin A showed excellent activity against multi-resistant Staphylococcus aureus strains (Zahner, 1999). In addition to the orthoesters, the terminal dichloroisoevernic acid residue is said to be essential for effectiveness (Wright, 1979). Like US Pat. No. 3,131,126, DE 1116864 describes the substance class of the avilamycins including a general reference to derivatives and the preparation and action of avilamycins.

Ziracin also belongs to the Orthosomycine group. Ziracin (SCH27899) is an evernimycin and has already been clinically tested.

Avilamycin A Ziracin

Both avilamycin A and ziracin have shown in practice, however, that the therapeutic use seems to be limited by the insufficient hydrophilicity.

Molecular cloning and characterization of the enzymes determining the biosynthesis of avilamycin A should be of great interest, especially for the class of orthosomycins and in particular for the avilamycins, since this information is the direction for the development could prescribe new (antimicrobial) antibiotics. The genes are an interesting system to study the formation and linkage of unusual deoxysugars and may therefore be of great value for combinatorial biosynthesis.

Previous work on the biosynthetic gene cluster of avilamycins led to the decoding of the sequence of an NDP-glucose synthase gene (aviD [serial number 53 according to Table 1]), an NDP-glucose-4,6-dehydratase gene (aviE1 [serial number 54 according to Table 1]) and a polyketide synthase gene (aviM (serial number 52 according to Table 1]). These presumably have a function as part of an iterative type I polyketide synthase for the formation of orsellic acid, an intermediate in the Biosynthesis of dichloroisoevernic acid: Expression of aviM in S. lividans led to the formation of orsellic acid [Gaisser, S., Trefzer, A., Stockert, S., Kirschning, A., & Bechthold, A. (1997), J Bacteriol. 179, 6271-6278].

In addition to finding and identifying suitable enzyme systems and synthetic routes, it was therefore an object of the invention to provide new antibiotics, in particular also those which have improved hydrophilicity.

Surprisingly, it was found that certain avilamycin derivatives not described in the prior art (also referred to as gavibamycins according to the invention) - in particular with a substitution pattern changed in crucial areas compared to avilamycin A - can solve this task and have both antibiotic activity and improved hydrophilicity demonstrate. The invention therefore relates to an avilamycin derivative according to general formula I, also in the form of its diastereomers or Enantiomers or racemic or other mixtures or pure diastereomers and / or enantiomers,

, where independently with the following exception

R1 is selected from H, COCH ₁ COC ₄ H ₉ , COCH (CH ₃ ) ₂ or

R2 is selected from H, CHO, COCH3 or CH (OH) CH ₃ ,

R3 corresponds to OCH ₃ ,

R4 corresponds to Cl, R5 corresponds to Cl,

R6 corresponds to CH ₃ ,

R7 corresponds to H, CH ₃ or CH ₂ OH,

R8 CH ₃ corresponds

and

R9 corresponds to CH ₃ ,

in which, in relation to at least one of the radicals R3-R6, R8 or R9 in formula I, in deviation from the above definition, the following applies:

R3 is to be replaced by OH,

R4 is to be replaced by H,

R5 is to be replaced by H,

R6 is to be replaced by H,

R8 is to be replaced by H.

and or

R9 is to be replaced by H, with the proviso that R1-R9 cannot simultaneously assume the meanings according to the respective combination in one of the compounds 1-4:

The expression "with the following exception" is to be understood to mean that there are exceptions to the general definitions of the radicals R1-R9 which immediately follow this expression, which have the phrase "in which at least one of the radicals R3-R6, R8 or R9 in Formula I deviating from the above definition, the following applies ".

In addition to their surprisingly strong antibiotic activity, the avilamycin derivatives according to the invention are distinguished in particular against Staphylococcus aureus, in particular also against one of the known orthosomycins such as avilamycin A or C and

Evernimycin (= everninomycin, = evernimicin) significantly improved

Hydrophilicity. It is precisely this increased hydrophilicity that makes these compounds attractive, especially antibiotic

Active ingredients because of increased hydrophilicity in certain therapeutic

Applications is very desirable. In addition, for this, as for all of the avilamycin derivatives according to the invention, which are also described below, it applies that it has a structure which is difficult to open up to classic organic synthesis. The underlying one

Use of a genetic engineering biosynthesis to produce the Avilamycin derivatives according to the invention thus open up modified, new and previously inaccessible active ingredients, in particular antibiotics.

An avilamycin derivative according to the invention in which at least R3 is to be replaced by OH is preferred in the context of this invention, with the proviso that R1-R9 cannot simultaneously take on the meanings according to the combination in compound 1:

Also preferred is an avilamycin derivative according to the invention in which at least R4 and R5 in formula I are to be replaced by H.

Also preferred is an avilamycin derivative according to the invention in which at least R6, R8 and / or R9 is / are to be replaced by H, with the proviso that R1-R9 do not simultaneously mean the meanings according to the combination in compound 3 or not simultaneously Can assume meanings according to the combination in compound 4:

It is also particularly preferred to combine these features, which leads to an avilamycin derivative according to the invention in which on the one hand at least R3 is to be replaced by OH and on the other hand at least R4 and R5 are to be replaced by H or at least R6, R8 and / or R9 is to be replaced by H.

A particularly preferred object of the invention that solves the problem in a particularly favorable manner is an avilamycin derivative according to general formula I, which is selected from compounds in which R1-R9 each have the meaning given in the table below, are combined as follows:

, preferably

The task is also solved by avilamycin derivatives, which can be produced by a special process that includes genetic engineering manipulations and biosynthesis. Another object of the invention is therefore an avilamycin derivative which is obtainable by the fact that, in a cultivable cell, the genes or enzymes required for the synthesis of an orthosomycin base body consisting of (a) a terminal dichloroisoevernic acid residue (A in formula I) and

(b) an heptasaccharide (B to H in formula I) esterified with it and linked via normal ester bond and ortho ester bond from:

(i) two D-olivose residues (B and C) (ii) a 2-deoxy-D-evalose residue (D), (iii) a D-fucose residue (E), (iv) a D- Mannose residue (F), (v) an L-lyxose residue (G) and (vi) a (methyl) eurekanate residue (H)

has, at least one nucleic acid, the sequence of which corresponds to at least 95%, preferably 97%, in particular exactly, the nucleic acid sequence according to one of Figures 1 to 54, is genetically modified, deleted and / or not expressed, the modified cell is cultivated, the Culture supernatant is obtained and worked up, the avilamycin derivative (s) is / are purified and isolated and, if appropriate, various derivatives are separated,

with the proviso that R1-R9 cannot simultaneously assume the meanings according to the respective combination in one of the compounds 1-16:

For the purposes of the invention, this means that "the cell has the necessary genes or enzymes for the synthesis of an orthosome base", that the genes coding for the necessary enzymes and / or the functional enzymes themselves are present in the cell are necessary for the synthesis of an “orthosomycin base body” from the precursors usually present. Examples would be the gene cluster according to the invention according to Fig. 109 or the "Open Reading Frames" (ORF) or genes according to sequence number 1-54 according to Table 1 in conjunction with Fig. 1 or the associated enzymes or proteins according to sequence number 55-108 according to Table 1 in conjunction with Fig. 1.

The definition of the “orthosomycin base body” has already been given, the arrangement of the ortho- and the normal ester bond being shown in the formula I. Avilamycins and evernimycins and avilamycin derivatives according to the invention include such a base body (see formula I). ,

For the purposes of this invention, gene is further understood to be a section of DNA from which a single mRNA molecule (which is then translated into a single polypeptide or protein) or a functional RNA molecule (rRNA, tRNA) is transcribed. For the purposes of this invention, “open reading frame” (ORF) is further understood to mean a DNA section that begins with a start codon, ends with an end codon and contains an uninterrupted sequence of codons for amino acids. The term “open reading Frame "(ORF) is used here to describe a cloned and sequenced DNA segment that corresponds to a gene.

Codon is the coding basic genetic unit. It consists of a triplet of three consecutive nucleotides that code for either an amino acid or the start or end of a polypeptide chain.

Furthermore, for the purposes of this invention, cells that can be cultivated are understood to mean cells that grow and multiply in vitro in a solid or liquid medium fed by a liquid or solidified nutrient solution, the culture medium. In the narrower sense, these are in particular cells from

Microorganisms or easily transfectable cells in which appropriate genes can be expressed. For example, gram-positive and gram-negative bacterial cells, e.g. Streptomyces cells (e.g. Streptomyces viridochromogenes Tu 57), but also systems such as mammalian cells, e.g. CHO cells (Chinese

Hamster ovary), or immortalized cell lines, e.g. HeLa- or HEK-

Cells, but also insect, fish, amphibian, mushroom or yeast cells etc.

For the purposes of this invention, a nucleic acid is understood to mean the basic unit of DNA and RNA and thus in particular the basic unit of a gene and an ORF. Correspondingly, a nucleic acid can comprise a gene or an ORF and a specific nucleic acid sequence (the sequence of the bases on the phosphate sugar). Backbone of a nucleic acid) accordingly define a gene or an ORF. Nucleic acid is also understood to mean sequences which, in addition to the coding regions, also contain further sequence regions, in particular at the 5 'or 3' end of the coding region. These sequences can have no function or can be promoter or enhancer signals, preferably bacterial signals or signals corresponding to the host cell system used for expression. In addition to the sequence regions coding for the proteins according to the invention, those nucleotide sequences which code for so-called “tags” (for example His or Flag tags) are very particularly preferred, so that the proteins according to the invention expressed in the host cells can be easily purified, for example by affinity chromatography Any nucleotide sequences which code for AS sequences and which contain a tag (for example an antigen) for binding to an antibody, for example on a column, can thus be attached to the coding nucleotide sequences according to the invention, preferably at the 5 'or 3' end The AS sequences which result from the combination of coding nucleic acids according to the invention with other nucleotide sequences are also disclosed.

For the purposes of the invention, genetic engineering means the use of various techniques with which DNA is introduced into a host cell or the DNA of a cell is specifically modified. This includes e.g. the use of cloning techniques, vectors, restriction enzymes etc.

Accordingly, genetically modified means that an intervention in the base sequence, the sequence that has changed the nucleic acid, in particular the base sequence has been shortened (up to the point of deletion) or mutations have been incorporated, usually with the result that the nucleic acid (the gene) does not or only does so can be transcribed into an mRNA. Deleted in this case means that a nucleic acid, which usually comprises a gene or an ORF, is completely or at least largely removed from the DNA, so that the nucleic acid (the gene) cannot be transcribed into an mRNA, or can only be modified. Accordingly, not expressed means that the nucleic acid has been changed in such a way that the nucleic acid (the gene) cannot be transcribed into an mRNA, or can only be changed, and accordingly the polypeptide or protein for which the nucleic acid (the gene or the ORF) originally coded.

Moderately stringent hybridization conditions are understood to mean varying standard conditions depending on the nucleic acid sequence used (oligonucleotide, longer fragment or complete sequence) or on the type of nucleic acid (DNA or RNA) used for the hybridization. For example, the melting temperatures for DNA: DNA hybrids are approx. 10 ° C lower than those of DNA: RNA hybrids of the same length. Under standard conditions, for example, depending on the nucleic acid, temperatures between 42 and 58 ° C in an aqueous buffer solution with a concentration between 0.1 to 5 x SSC (1 X SSC = 0.15 M NaCl, 15 mM sodium citrate, pH 7.2 ) or additionally in the presence of 50% formamide, such as 42 ° C in 5 x SSC, 50% formamide. The hybridization conditions for DNA: DNA hybrids are advantageously 0.1 × SSC and temperatures between approximately 20 ° C. to 45 ° C., preferably between approximately 30 ° C. to 45 ° C. For DNA: RNA hybrids, the hybridization conditions are advantageously 0.1 × SSC and temperatures between approximately 30 ° C. to 55 ° C., preferably between approximately 45 ° C. to 55 ° C. These specified temperatures for the hybridization are, for example, calculated melting temperature values for a nucleic acid with a length of approx. 100 nucleotides and a G + C content of 50% in the absence of formamide. The experimental conditions for the DNA hybridization is found in relevant textbooks on genetics, such as in Sambrook et al. ("Molecular Cloning", Cold Spring Harbor Laboratory, 1989), and can be calculated according to formulas known to the person skilled in the art, for example depending on the length of the nucleic acids, the type of hybrid or the G + C content. The person skilled in the art can obtain further information on hybridization from the following textbooks: Ausübel et al. (eds), 1985, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Harnes and Higgins (eds), 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press at Oxford University Press, Oxford; Brown (ed), 1991, Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford.

For the purposes of this invention, cultivation is understood to mean the in vitro cultivation of cultivable cells, as a result of which they grow and multiply, nourished in a solid or liquid medium by a liquid or solidified nutrient solution, the culture medium.

Culture supernatant is understood to mean the liquid culture medium which, in addition to the nutrients for the cells which can be cultivated, also contains the metabolites and substances (e.g. avilamycin derivatives) released into the medium by them. This culture supernatant can be obtained and worked up, which means in particular the suctioning off of the supernatant and / or a filtration with which the solids remaining from the cultivation and the cells are separated off.

The culture supernatant, which in the context of this invention mostly contains avilamycin derivatives according to the invention, can be purified after working up, including, for example, chromatographic separation and / or separation via liquid phases or a combination of these procedures is to be understood. Examples are a solid phase extraction with a methanol-in-water gradient or an ethyl acetate extraction. The fraction containing the avilamycin derivatives is separated as much as possible from other fractions containing other constituents of the culture supernatant and the avilamycin derivatives are thus largely isolated. However, the method of salting out or recrystallization or recrystallization can also be considered as alternative separation and / or purification methods. If necessary, isolation and separation of the individual derivatives can then follow, in particular chromatography methods being used here. Preparative HPLC methods or affinity chromatography methods are very particularly preferred.

It is preferred if, in the production process by which the avilamycin derivative according to the invention is defined, the cultivable cell is selected from a cell of the Streptomyces viridochromogenes type or a cell which, with the exception of the genetically modified, deleted or unexpressed nucleic acid / s, the nucleic acids 1 to 54 according to Table 1 in conjunction with Fig. 1 or at least 95%, preferably 97%, of homologous nucleic acids or hybridizes with one of these sequences under moderately stringent conditions or contains the gene cluster according to Fig. 109. The second point of the selection is to be understood in particular as cells in which the enzymes necessary for avilamycin derivative synthesis are expressed by genetic engineering methods, one of the nucleic acids coding for an enzyme coding for endogenously occurring in the Streptomyces viridochromogenes Tu 57 or is deleted or is not expressed, in particular the nucleic acid / DNA is not even introduced into the host cell by genetic engineering. However, it is particularly preferred if the Cell is selected from a cell of the type Streptomyces viridochromogenes, in particular a cell of the type Streptomyces viridochromogenes Tu 57 or A 23575.

In any case, it is preferred if in the process the modified (e.g. deleted or not introduced into the host cell) nucleic acid (s) for a methyl transferase and / or for a halogenase encoded.

Alternatively, the production can also take place outside of an in vivo process as in vitro synthesis. The enzymes and / or enzyme systems required for the synthesis are specified in at least one test batch, the reaction steps required for the synthesis preferably being carried out catalytically by the enzymes required and according to the invention in several test batches connected in series. Possibly. Separation and / or purification steps for purifying the desired intermediate products can be inserted between the individual reactions carried out in a suitable sequence.

Methyl transferases are understood to be enzymes that can transfer a methyl group to an organic molecule. In the sense of the invention, these are in particular enzymes which transfer a methyl group to either orsellic acid or to the sugars, preferably after the formation of the heptasaccharide, in particular the ORFs aviG2, avι ^' G3, aviG5, aviG6, aviG1, aviG4, aviRa and aviRb, especially aviG4.

Halogenases are understood to be enzymes that can transfer halogens enzymatically to organic molecules. For the purposes of the invention, these are in particular enzymes which relate to orsellic acid one, preferably two Transfer Cl residues at positions R4 and / or R5, especially the ORF aviH.

Accordingly, it is a particularly preferred object of the invention if, with respect to the above-mentioned production method, the sequences of the changed nucleic acid (s) before the change are at least 95%, preferably 97%, in particular exactly, the nucleic acid sequence (s) at least one of the sequences according to serial No. 1 or 2-7 according to Table 1 in conjunction Fig. 1, preferably one of the sequences with sequence numbers 1, 2, 4 or 6 (Table 1 in connection with Fig. 1) corresponds to en, in particular the sequence with sequence number 2 or the sequences with sequence numbers 2 and no. 1, No. 2 and No. 4 or No. 2 and No. 6 (according to Table 1 in connection with Fig. 1) or hybridized with one of these sequences under moderately stringent conditions.

For the purposes of this invention, “before the change” means that the modified nucleic acid before the genetic engineering manipulation of it, ie before the deletion or the change, in particular shortening or mutation in the base sequence, but also before the step, even this nucleic acid / DNA not first to be introduced into the host cell by genetic engineering, which has the nucleic acid sequence mentioned.

It is further preferred if, in the production process defining the avilamycin derivatives, the change in the nucleic acid (s) leads to the fact that the protein (s) or polypeptide (s) encoded by the genetically modified nucleic acid (s) or polypeptide (s) after the genetic engineering change is no longer synthesized.

Polypeptide is understood to be a peptide with between 10 <and <100 amino acid residues and a protein is a macromolecule with more than 100 amino acid residues linked via peptide bonds. there the proteins in the context of this invention are preferably enzymes. Of course, other proteins within the meaning of this invention also fall under this term.

The avilamycin derivatives according to the invention described so far have predominantly or all the advantages compared to the related orthosomycins described in the prior art, in particular compared to avilamycin A, of being more hydrophilic, which offers considerable therapeutic advantages. This applies in particular to a comparison with the Avilamycin A or C or also with the Evernimycin Ziracin.

It was also an object of the invention - in addition to the provision of new antibiotics - to clarify the biosynthesis of avilamycin A in order to develop new antimicrobial substances or new processes for their production based thereon. A key point was the molecular cloning and the characterization of the genes involved in avilamycin biosynthesis. An approximately 60 kB piece was sequenced around the well-known genes aviD, aviEl and aviM. It turned out that the genes involved were arranged in close proximity to one another in a cluster. The sequence of the individual ORFs and their arrangement on the central gene cluster (consecutive numbers 1 to 54) are shown in Fig. 1 in connection with Table 1, respectively Fig. 109. As already stated, the sequence was an NDP-glucose synthase gene (aviD [serial number 53 according to Table 1 in conjunction with Fig. 1]), an NDP-glucose-4,6-dehydratase gene (aviEl [serial number 54 according to Table 1 in connection with Fig. 1]) and a polyketide synthase gene (aviM [serial number 52 according to Table 1 in connection with Fig. 1]) as well as their presumed function as part of an iterative type I polyketide synthase for the formation of Orsellic acid, an intermediate in the biosynthesis of dichloroisoevernic acid [Gaisser, S., Trefzer, A., Stockert, S., Kirschning, A., & Bechthold, A. (1997), J Bacteriol. 179, 6271-6278]. The sequences discovered by the extensive cloning of the remaining ORFs involved in the synthesis of the avilamycins can also be seen in Fig. 1, as can the relative arrangement on the gene cluster in Fig. 109. Here, the specification of the serial number from Table 1 allows the assignment to the name of the ORFs. The sequences in Fig. 1 can be found under the name by name, in the manner shown in the description of Fig. 1. The exact cloning strategy and further details of the sequencing are shown in the examples as well as the functional analysis and characterization of the genes found (ORF's). The assignment of the ORF abbreviation to function and sequence (including the derived protein sequence) can be found in Table 1 following the illustration description.

Another important subject of the invention is therefore one (or more) nucleic acid (s) which in their sequence are at least 95%, preferably 97%, in particular exactly, the nucleic acid sequence according to one of the sequences of the sequence numbers 1 to 51 according to Table 1 in conjunction Fig. 1 corresponds / correspond or hybridized with one of these sequences under moderately stringent conditions. In particular, sequences with sequence numbers 48 and 49 (according to Table 1 and sequence representation in Fig. 1) with function as rRNA methyltransferases (aviRa and aviRb) and also the sequences with sequence numbers 50 and 51 (according to Table 1 iVm Fig. 1) with function as ABC transporter genes (aviABCI and aviABC2) that mediate resistance to avilamycins, or sequences that correspond to at least 95% of these sequences with the aforementioned serial numbers or with one of these sequences below hybridize moderately stringent conditions, described in the present invention. Incidentally, also mixtures of nucleic acids, any subcombinations of the 1 with the current numbers 1 to 51 from table 1, for example. Mixtures of two, three, four, ..., 50 nucleic acids in any combination are also disclosed according to the invention, if appropriate also as a combination on a nucleic acid strand or on different strands.

Particular preference is given to (a) nucleic acid (s) which comprise at least 95%, preferably 97%, in particular exactly, the nucleic acid sequence according to one of the sequences with the current numbers 1 to 32 according to Table 1 (in conjunction with Fig. 1), preferably 1 to 7, in particular 1, 2, 4 or 6, or one of the sequences with the sequence numbers 48 to 51 or 43, 44 or 46 according to Table 1 (in conjunction with Fig. 1) or which corresponds with one of these Sequences hybridized under moderately stringent conditions.

Another object of the invention are also gene clusters which contain "open reading frames", preferably 54, which in their nucleic acid sequence are at least 95%, preferably 97%, in particular exactly, the nucleic acid sequences according to the sequences with the sequence numbers 1 to 54 (Table 1 in connection with Fig. 1) or hybridize with one of these sequences under moderately stringent conditions and which are arranged on one strand of nucleic acid or in any combination on one or the other strand, preferably according to Fig. 109. The genes in an invention Gene clusters can contain 2, three, four, ..., 54 genes according to the invention, optionally in combination with the already known genes, in any strand distribution and sub-combination, in particular the sections between the ORFs can be any nucleotide sequence. This means in particular a gene cluster according to Fig. 109, but also gene clusters that contain the corresponding nucleic acids, possibly also in a different arrangement, whereby it is preferred - but not necessary - that all ORFs according to the order numbers 1-54 (Table 1 in conjunction with Fig. 1) can be found in the gene cluster.

For the purposes of this invention, the term gene cluster is understood to mean a section of DNA on which several genes are located in close spatial proximity. Such gene clusters according to the invention can be present in a vector, for example a BAC or YAC, a cosmid or plasmid. Vectors which contain at least one sequence according to the invention are thus also the subject of the present invention. Genes according to the invention can be combined in vectors according to the invention with further signal sequences or further genes, in particular further antibiotic resistance genes.

Protein and polypeptide sequences could be derived from the newly discovered sequences of the ORFs or genes. Accordingly, the invention further relates to a protein or polypeptide which has at least 95%, preferably 97%, in particular exactly, the amino acid sequence of the amino acid sequence according to one of the sequences with the current numbers 55-101 (Table 1 in conjunction with Fig. 1). equivalent.

It is preferred if the protein or polypeptide according to the invention is at least 95%, preferably 97%, in particular exactly, the nucleic acid sequence according to one of the sequences with the sequence numbers 55 to 86 or 97, 98 or 100 or 102 to 105 (Table 1 in conjunction with Fig. 1), preferably 55 to 61, in particular 55, 56, 58 or 60. Another subject is accordingly also a protein or polypeptide which is encoded by a nucleic acid according to one of claims 12 or 13. In the context of this invention, “coding” means that the codons (see above) of the corresponding nucleic acid section (gene or ORF) code for the corresponding amino acid sequence, that is to say after transcription and translation, a corresponding protein or polypeptide with this amino acid sequence is formed.

In particular, the proteins according to the invention are enzymes or part of a multienzyme complex. Of course, they can also have other functions.

Since, on the one hand, the avilamycin derivatives according to the invention are defined or are produced by a genetic engineering or biotechnological process, on the other hand, the newly discovered genes or proteins (enzymes) can be used in gene or biotechnological processes for the production of corresponding antibiotics , In the context of this invention, genetically modified cells almost inevitably have an important function.

Another subject of this invention is therefore genetically modified cells containing at least one non-endogenous nucleic acid according to the invention, a non-endogenous gene cluster according to the invention and / or a non-endogenous protein or polypeptide according to the invention.

Likewise, a cell is a further subject of the invention, the at least one genetically modified nucleic acid, the sequence of which before the change is at least 95%, preferably 97%, in particular exactly, the nucleic acid sequence according to one of the Sequences with the running numbers 1 to 54 (Table 1 in connection with Fig.1) corresponded to or which hybridized with one of these sequences under moderately stringent conditions.

A particularly preferred object of the invention is a cell of the Streptomyces viridochromogenes type, preferably of the Tü57 subtype, in which at least one of the nucleic acids has been genetically modified or deleted with a sequence with one of the current numbers 1-54 (Table 1 in conjunction with Fig. 1) , It is particularly preferred if in the corresponding cell at least one of the nucleic acids with a sequence with sequence number 1 or 2-7 (Table 1 in conjunction with Fig. 1), preferably with one of the sequences with sequence number 1, 2, 4 or 6 (Table 1 in connection with Fig. 1), in particular with a sequence with consecutive No. 2 or with sequences with the consecutive No. 2 and No. 1, No. 2 and No. 4 or No. 2 and No. 6 ( according to Table 1 in connection with Fig. 1) has been genetically modified or deleted.

It is also particularly preferred if the cell is of the mutant type Streptomyces viridochromogenes GW4, Streptomyces viridochromogenes, GW4-AM1, Streptomyces viridochromogenes GW2 or Streptomyces viridochromogenes GW5, with the mutant type Streptomyces viridochromogenes GW4 being Avilamycin derivatives in which derivatives are synthesized = OH, the mutant type Streptomyces viridochromogenic GW4-AM1 avilamycin derivatives in which R3 = OH, R4 = H and R5 = H are synthesized, the mutant type Streptomyces viridochromogenic GW2 avilamycin derivatives in which R3 = OH and R6 = H and the mutant type Streptomyces viridochromogenes GW5 avilamycin derivatives are synthesized in which R3 = OH and R9 = H. According to the above, another object of the invention is the use of a nucleic acid according to the invention, a gene cluster according to the invention, a protein or polypeptide according to the invention and / or one of the cells according to the invention for producing an avilamycin derivative, preferably an avilamycin derivative according to the invention.

Another object of the invention is a process for the preparation of avilamycin derivatives according to the invention with the following steps:

(1) in a cultivable cell which consists of the genes or enzymes required for the synthesis of the orthosomycin base body

(a) a terminal dichloroisoevernic acid residue (A in formula I) and

(i) two D-olivose residues (B and C)

(ii) a 2-deoxy-D-evalose residue (D),

(iii) a D-fucose residue (E),

(iv) a D-mannose residue (F),

(v) an L-Lyxose residue (G) and (vi) a (methyl) Eurekanat residue (H)

has, at least one nucleic acid, the sequence of which is at least 95%, preferably 97%, in particular exactly, the nucleic acid sequence with one of the current numbers 1 to 54 in accordance with Table 1 in conjunction. Fig. 1 corresponds or one

Nucleic acid with one of these sequences under moderate stringent conditions hybridized, genetically modified, deleted or not expressed,

(2) the cell modified in this way is cultivated, (3) the culture supernatant is obtained,

(4) the culture supernatant is worked up and the avilamycin derivative (s) formed is purified and isolated,

(5) if necessary, different derivatives are separated.

It is preferred if, in this method, the culturable cell is selected from a cell of the Streptomyces viridochromogenes type or a cell which, with the exception of the genetically modified, deleted or unexpressed nucleic acid, contains the nucleic acids according to serial numbers 1-54 according to Table 1 in conjunction. Fig. 1 or at least 95%, preferably 97%, of homologous nucleic acids or sequences hybridizing with these sequences or contains the gene cluster according to the invention. The latter is referred to in the specialist literature as heterologous expression. It is particularly preferred if the cell is selected from a cell of the type Streptomyces viridochromogenes, Streptomyces lividans, Streptomyces albus or Streptomyces fradiae, in particular a cell of the type Streptomyces viridochromogenes Tu 57 or A 23575.

An alternative procedure is also possible according to the invention. However, after carrying out process steps (1) and (2), the avilamycin derivative is not obtained from the culture supernatant, but rather accumulates in the host cells. According to the alternative method, the host cells are therefore harvested in method step (3), subsequently digested and the avilamycin derivatives separated from the other cell components and finally purified. For the separation and purification, the abovementioned and all processes known to the person skilled in the art can be used.

It is further preferred if, in the method according to the invention, the changed nucleic acid (s) codes for a methyl transferase and / or for a halogenase. It is particularly preferred if the sequence (s) of the changed nucleic acid (s) before the change is at least 95%, preferably 97%, in particular exactly, the nucleic acid sequence (s) of at least one of the sequences with the current number 1 or 2-7 according to Table 1 in conjunction Fig. 1, preferably a sequence with the running numbers 1, 2, 4 or 6 (Table 1 in connection with Fig. 1), corresponds to / s, in particular the sequence with the running number 2 or the sequences with the running numbers 2 and No. 1, No. 2 and No. 4 or No. 2 and No. 6 (according to Table 1 in connection with Fig. 1).

It is further preferred if, in the method according to the invention, the change in the nucleic acid (s), in particular methyl transferases and / or halogenases, leads to the fact that the protein (s) or polypeptide (s) encoded by the genetically modified nucleic acid (s) / e is no longer synthesized after the genetic engineering change.

The avilamycin derivatives according to the invention are in principle toxicologically harmless, so that they are suitable as active pharmaceutical ingredients in pharmaceuticals. The invention therefore furthermore relates to medicaments comprising at least one avilamycin derivative according to the invention, preferably at least two, in particular also mixtures of one or more avilamycin derivatives with at least one further antibiotic from the prior art, for example vancomycin, penicillin, streptomycin, neomycin , Kanamycin, Sisomycin, Amikacin and / or Tobramycin, as well as suitable additive and / or Excipients. Other bacteriostatic or bactericidal substances can also be combined with substances according to the invention, for example cephalosporins, chloramphenicol, ethambutol, cephalosporins, isonicotinamides, tetracyclines, sulfonamides, oxalactams (for example flomoxef, clavulanic acid) and / or nitrofurans.

This also includes carrier materials, fillers, solvents, diluents, dyes and / or binders. The pharmaceuticals can be administered as liquid dosage forms in the form of injection solutions, drops or juices, as semi-solid dosage forms in the form of granules, tablets, pellets, patches, capsules, plasters or aerosols. The choice of excipients etc. and the amounts to be used depend on whether the medicinal product is orally, orally, parenterally, intravenously, intraperitoneally, intradermally, intramuscularly, intranasally, buccally, rectally or locally, for example on the skin, mucous membranes or in the eyes to be applied. Preparations in the form of tablets, dragees, capsules, granules, drops, juices and syrups are suitable for oral administration, and solutions, suspensions, easily reconstitutable dry preparations and sprays are suitable for parenteral, topical and inhalative administration.

Formulations which can be used orally or percutaneously can release the avilamycin derivatives according to the invention with a delay and thus achieve a more uniform plasma level. In principle, other active substances known to the person skilled in the art can be added to the medicaments according to the invention.

The amount of active ingredient to be administered to the patient varies depending on the weight of the patient, the type of application, the Indication and the severity of the disease. Usually 0.005 to 1000 mg / kg, preferably 0.05 to 5 mg / kg, of at least one avilamycin derivative according to the invention are applied.

Since an antibiotic effect has been demonstrated for the avilamycin derivatives according to the invention, they are of course in principle suitable for the treatment of diseases, in particular for the treatment of infectious diseases, or for the manufacture of a medicament for the treatment of such diseases. Another object of the invention is accordingly the use of an avilamycin derivative according to the invention for the manufacture of a medicament with an antibiotic effect for the treatment of, for example, infectious diseases. Infectious diseases are understood to mean diseases which are based on an infection with a viral, a bacterial or a protozoological pathogen. The present antibiotics according to the invention are therefore also suitable for the treatment of mycoses, in particular cutaneous and subcutaneous mycoses.

However, the avilamycin derivatives according to the invention are preferably used to combat bacterial infections. In particular are

Infections with the following pathogens: leprosy bacteria,

Mycobacteria, Neisseries, tuberculosis bacteria, actinomycetes,

Corynebacteria, listeria, clostridia, bacilli, enterococci,

Streptococci, staphylococci, in particular also for the treatment of infections with Staphylococcus aureus strains, rickettsiae,

Chlamydia, mycoplasma, borrelia, spirochetes, brucella,

Borderellen, pseudomonas, Helicobacter, hemophilus, vibrions,

Shigellen, Yersinia, Salmonellen and others among the family of

Enterobacteriaceae falling representatives. Accordingly, the substances according to the invention are used to treat all clinical

Disease patterns caused by, for example, the aforementioned bacterial strains caused. The following clinical pictures may be mentioned as examples: tuberculosis; pneumonia; Typhus; Paratyphoid; Syphilis; Gastritis; Gastroenteritis; Ruhr; Pest; enteritis; extraintestinal infections, peritonitis and appendicitis with E. coli and intestinal infections with EHEC, EPEC, ETEC or EIEC; Cholera, legionnaires' disease, whooping cough, brucellosis, Lyme disease, leptospirosis, typhus, trachoma, gonorrhea, meningitis, septicemia, leprosy etc.

Another object of the method is also the treatment of a person or animal who needs this treatment with an avilamycin derivative according to the invention, preferably in the case of infectious diseases, in particular with the participation of Staphylococcus aureus.

In the following section, the invention is further illustrated by examples, without being restricted thereto.

Examples and illustrations:

pictures:

Figure 1 shows the sequence of the entire gene cluster with its 54 nucleic acid sequences of the ORFs from Streptomyces viridochromogenes Tu 57. Figure 1 contains the short names of the corresponding nucleic acid sequences, these short names (without the prefix "Avi") being inserted at the lines that have the start codons of the 54 sequences, the AS encoded by the respective start codon is circled in. The arrow drawn in at these points shows the reading direction (backwards or forwards) of the genes with the start codon as the starting point. Fig. 1 contains the nucleotide sequences of the two complementary DNA strands as well as the (partly fictitious) AS sequences for both strands in all three reading frames, thus a total of 2 nucleotide sequences and the 6 protein sequences potentially resulting therefrom (one-letter code) , The three protein sequences of the upper nucleotide strand are shown above the associated nucleotide sequence, the three protein sequences of the lower complementary nucleotide sequence below the associated lower nucleotide strand. The 54 protein sequences in the gene cluster shown by name in Fig. 1 result from Fig. 1 by choosing a circled AS as the starting point and then in this reading frame, ie in the corresponding line (for example, 2nd line below the lower nucleotide sequence), the AS sequence is read in the direction indicated by the arrows, i.e. in the following either forwards or backwards. The sequence ends with the stop codon in the corresponding reading frame, with stop codons being marked by an “asterisk” symbol in the corresponding line.

The nucleotide sequence belonging to the ASF of an ORF results from the corresponding triplet located above or below (for the upper strand). The one-letter designation of the amino acid is arranged in such a way that it lies above or below the middle nucleotide of the codon coding for this AS.

In the table below, the names of the 54 coding regions in the gene cluster are assigned sequential numbers, the sequential numbers 1 to 54 indicating the nucleotide sequences and the sequential numbers 55 to 108 corresponding to the associated AS sequences, specifically the nucleotide sequence with the serial number 1 for the AS with the serial number 55, the nucleotide sequence with serial number 2 for the AS with serial number 56 etc.

Figure 109 shows the relative arrangement of the ORFs found on the gene cluster.

Figure 110 shows a Southern blot with the mutant S. viridochromogenes GW4.

Figure 111 shows the mass spectrum of the products of mutant S. viridochromogenes GW4

Figure 112 shows the mass spectrum of the hydrolyzed products of mutant S. viridochromogenes GW4.

The assignment of the ORF abbreviations to their function and sequence (including the derived protein sequence) can be found in Table 1 below.

Sheetl:

Examples

5 Example 1

General methods and materials:

a)

Bacterial strains, plasmids and culture conditions.

10

Streptomyces viridochromogenes Tü57 was cultivated with 1% malt extract, 0.4% yeast extract, 0.4% glucose and 1 mM CaCl ₂ , at a pH of 7.2 (HA medium) at 37 ° C. Streptomyces viridochromogenes Tü57 and all mutants in NL19 + - were used to produce avilamycin A

15 medium, which contained 2% D-mannitol, 2% soy flour and 20 mM L-valine and was adjusted to pH 7.5, was cultured. For DNA manipulation Escherichia coli XL-1 Blue MRF '(Stratagene) used as the host cell. Before the transformation of S. viridochromogenes Tü57, the plasmids in E. coli ET 12567 (dam ~, dcm ~, hsdS, Cm ^R ) was attracted to obtain unmethylated DNA. E. coli strains were cultured on Luria-Bertani (LB) agar or liquid medium containing the appropriate antibiotic.

b) General genetic engineering manipulations. PCR and DNA sequencing / sequence analysis

Standard methods of molecular biology - as known to the person skilled in the art - were carried out. The isolation of E. coli plasmid DNA, DNA restriction, DNA modification such as the “filling-in sticky ends” and the “Southern” hybridization were carried out in accordance with the protocols of the manufacturers of the kits, enzymes and reagents (Amersham-Pharmacia, Boehringer Mannheim , Promega, Stratagene). Streptomyces protoplast formation, transformation, and regeneration were carried out as usual. The PCR was carried out with a Perkin Elmer GeneAmp 2400 thermal cycler, the conditions being as described and usual. The oligonucleotide primers used were:

AviG4F (δ'-GGACGCCTATCTGTGCCACCCCTTCCTGGT-S '), AviG4R (5, -TGAGCGCTCGCCTAGACAGAATCATCTCCC3'), S2A (5'-GCGTCCATCTTGCCGGGA-3 ') and S2B (5, -CGGGGGAT.

The nucleotide sequencing was carried out with the dideoxy Chain termination method was carried out using an automatic laser fluorescence sequencer (Perkin Elmer ABI). The sequencing reactions were carried out using a thermosequenase cycle sequencing kit with 7-deaza-dGTP (Amersham) and standard primers (M 13 universal and reverse, T3, T7). With the DNASIS software package (version 2.1, 1995; Hitachi Software Engineering), a computer-aided sequence analysis and the database search with the BLAST 2.0 program were carried out on the server of the National Center for Biotechnology Information, Bethesda MD, USA. The sequences presented are stored in the "GenBank" database under the access number ("Accession Number") AF333038.

c)

Construction of a gene inactivating plasmid

aviG4: A unique / Vcol restriction site in the aviG4 gene (running No. 2, Fig. 1), which is located on the 1.9 fragment, which is ligated into the SacI and EcoR \ interfaces of pBSK, was used for the targeted inactivation selected to move the reading frame. The 1.9 kb fragment was digested with Sacl and Kpn \ and was ligated into the gene inactivation plasmid pSP 1. After restriction digestion with Λ / col, treatment with the Klenow fragment of E. coli DNA polymerase I and re-ligation, the intended change was confirmed by DNA sequencing. The plasmid formed was named pMIKG4E3.

avi i: The unique Naή interface in the av / H gene, which is ligated to the 3.7 kb Sacl fragment in pBSK-, was identified by Naή-

Restriction digestion and subsequent treatment as described for aviG4 changed. The sequencing of different plasmids showed the correct change. The 3.7 kb fragment was cloned into pSP1 to generate the gene inactivation plasmid pSP 1S2Na.

d) Analysis of new avilamycin derivatives DC analysis

Streptomyces viridochromogenes Tü57 and the mutants GW-4 and GW4-AM1 were incubated for three days. The cultures were filtered off and the filtrate was applied to a solid phase extraction cartridge (SepPakCiβ, Waters). The cartridge was eluted with a gradient between 10% and 100% methanol in water. Avilamycin derivatives elute with the fraction containing 60-70% methanol. After extraction with ethyl acetate and removal of the solvent, the avilamycin derivatives were redissolved in methanol and by TLC on silica gel plates (silica gel 60 F254, Merck) with methylene chloride / methanol (9: 1, v / v) as solvent measured. Avilamycin derivatives had been detected after treatment with anisaldehyde / H ₂ S0 ₄ .

e)

HPLC-UV analysis

Analytical HPLC-UV was carried out on a Hewlett Packard 1090 liquid chromatograph with a photodiode array detector and an HP-ODS-Hypersil 5Mm, 200 x 2 mm column. The sequence of the solutions was as follows: solution A, 0.04M (NH ₄ ) HPO pH 7.0 buffer; Solution B, 100% methanol; a non-linear gradient, with 30-62% of solution B distributed over 25 min at a flow rate of 0.2 ml / min. f) HPLC-MS analysis

For the HPLC-MS analysis, the avilamycin derivatives were on an HPLC system (HP 1110, Hewlett-Packard, Waldbronn) with an HP ODS Hypersil Cι ₈ column (2.1 by 100 mm; 5 μm) at a flow rate of 0.1 ml / min, detection run at 220 nm and the following gradient: 0-5 min from 0% to 20% B, 5.1-120 min to 90% B (solution A, H ₂ O: MeOH 3: 2; solution B, MeOH). Mass spectra were recorded on a Bruker Esquire-LC 1.6n mass spectrometer (Bruker Daltonik, Bremen) with an electrospray (ES) ion source (positive ion mode). The measuring width was between 200 - 1800 m / z.

g) GC-MS analysis

An analysis of the new gavibamycin derivatives (avilamycin derivatives according to the invention), which had been synthesized by the mutated cell Streptomyces viridochromogenes GW4, was carried out after etylation with GC-MS analysis. The derivatives were dissolved in a mixture of DMSO and acetonitrile (3:40). After adding ethyl iodide and K ₂ C0 ₃ , the reaction proceeded overnight. After the solvent had been stripped off, the derivatives were hydrolyzed with HCl / methanol at 115 ° C. for 15 min. After the solvent had been stripped off, the derivatives were extracted with diethyl ether and analyzed by GC-MS. A Hewlett Packard 5973 MSD system was used to generate El (electron impact) spectra (column: SE54, 12m x 0.25mm; d _f = 0.125μ). The column temperature was programmed as follows: 50 ° C for 2 min; 25 ° C / min to 100 ° C; 5 ° C / min to 250 ° C. Example 2

Cloning and sequencing of the avilamycin cluster

A 60kb section of the chromosome of the S. viridochromogenes Tü57, which contains genes that are involved in the biosynthesis of avilamycins, was cloned and sequenced. An analysis of the DNA sequence revealed 54 "open reading frames". It was already known that the NDP-glucose 4,6-dehydratase gene aviE and the orsellinic acid synthase gene aviM are essential for the biosynthesis of avilamycin A DNA flanking, isolating and sequencing the aviE and a ^' M genes to identify the avilamycin biosynthetic gene cluster, 17.6 kb upstream from aviM and 35.9 kb downstream from aviE were sequenced. The sequenced genes and their function are shown in Table 1. The genetic arrangement of the biosynthetic avilamycin gene cluster is shown in Fig. 109. The cluster is flanked by an avilamycin resistance gene (aviRb) and a deoxy sugar synthesis gene (avι ^' Z2) Sections are 25 genes (aviXW-aviGT4), all of which are transcribed in the same direction.

Example 3 Analysis of ORF's

a) General A computer analysis of the found sequences of the ORFs was carried out. The results of a sequence comparison were predominantly associated with the knowledge of the biosynthesis of avilamycin A. The results of these considerations based on the present experiments are shown in Table 1 read.

In the following, the functional considerations are presented using selected examples, especially the methyl transferases and halogenases.

b)

Genes with a function in the biosynthesis of

dichloroisoeverninic acid

AviM is responsible for the formation of orsellic acid during avilamycin biosynthesis. AviN, which is upstream from aviM, should code for an enzyme that controls the start signal for the synthesis of orsellic acid. The biosynthesis of dichloroisoevernic acid (A in formula I) starting from orsellic acid requires methylation and di-halogenation. It was surprisingly found that AviG4, the DmpM, resembles an O-demethylpuromycin-O-methyltransferase from S. alboniger (44% identical AS), and AviH, the PltA, a halogenase from Pseudomonas fluorescens Pf-5, which is involved in pyoluteorin biosynthesis ( 39% identical AS) is involved, are responsible for the modification of orsellic acid.

c)

Genes with a role in the biosynthesis of deoxy sugars.

2-deoxy-D-evalose differs from D-olivose in a methyl group at the C3 position. It is believed that dNDP-4-keto-2,6-dideoxy-D-glucose is an important intermediate in the biosynthesis of this methylated deoxy sugar. Methylation by AviG1, which is similar to TylCIII, a 3C-methyltransferase from S. fradiae (54% identical AS), and ketoreduction by either AviZ1 or AviZ2, which are similar to both ketoreductases and oxidoreductases, complete the biosynthesis.

d)

Genes with a function in the modification of the heptasaccharide chain

In addition to aviGI, aviG4, aviRa and aviRb, four other methyltransferase genes were found in the cluster (aviG2, aviG3, aviG5 and aviG6). They have been identified as potential methyltransferase genes either by the fact that their product resembles methyltransferases from other organisms, or by the presence of motifs typically found in various methylating proteins. Avilamycin derivatives which did not contain a methyl group at different positions in the molecule were produced from a cell line according to the invention. This indicates that the methylation takes place at a very late point in the biosynthesis. AviG2, AviG3, AviG5 and AviG6 should methylate on the D-fucose residue (E), D-mannose residue (F) and on the methyl-eurekanate residue (H) of avilamycin A.

Example 4

Production of an avι ^' G4 gene substitution mutant

To inactivate aviG4, the plasmid pMIKG4E3 was constructed (see Example 1) in order to allow the wild-type gene to be replaced by a mutated allele. After protoplasts were formed and S. viridochromogenes was transformed with the plasmid pMIKG4E3, erythromycin-resistant colonies were obtained. The transformation effectiveness was approximately 10 colonies per µg of plasmid DNA. Several colonies were left without Erythromycin cultured on plates to select after loss of resistance. Different sensitive colonies were obtained, which indicates that it is a result of a "double cross-over". Two mutants, G4 / 24/20 and G4 / 24/30, were further investigated. PCR fragments which were found under Use of the primers aviG4F and avι ^' G4R DNA from G4 / 24/20 and G4 / 24/30 could not be cut by Nco \, while PCR fragments from wild-type DNA could be cut by this enzyme To detect the deletion in aviG4, Southem blot analyzes were carried out as follows: Ncol-cut, chromosomal DNA was obtained from G4 / 24/20 and G4 / 24 / 30. When this DNA with a 1.9 kb fragment that covered the whole aw ^' G4 gene was hybridized, an 11 kb fragment was detected, while the expected 5 kb and 6 kb fragments were found in the S. viridochromogenes Tü57 line (Fig. 110). The mutant G4 / 24 / 30 was used under the new name S. viridochromogenes GW4 for further experiments.

Example 5

Production of an av / G4 ~ aviH double gene replacement mutant

The plasmid pSP 1 S2Nar was developed to switch off the aw ^' H gene (see Example 1). S. viridochromogenes GW4 protoplasts were transformed with this plasmid. There were approximately 20 erythromycin-resistant colonies per μg DNA. Some of them were cultured to screen for erythromycin resistance loss (indicating a "double cross-over"). The mutant GW4-AM1 was selected for further experimentation. A 1.34 kb PCR fragment that was generated using the Primer S2A and S2B obtained from H / 3/16 could not be cut by Narl while the PCR fragment from GW4 was digested by the enzyme, and a was used to detect the deletion in aviH Southern blot analysis performed. Chromosomal DNA from H / 3/16 was cut with Naή and hybridized with a 3.7 kb probe containing the aviH gene. A 5.7 kb fragment was detected, whereas in the case of chromosomal DNA from GW4, the fragments were, as expected, 4.3 kb and 1.4 kb (not shown).

Example 6

Completion of S. viridochromogenes GW4 and S. viridochromogenes GW4-AM1

To clearly check whether the mutation only affects the desired and no other genes, av \ ^' G4 and aviH were ligated behind the ermE-up promoter, cloned into the integration plasmid pSET152 and introduced into the corresponding mutants by protoplast transformation. The production of avilamycins and gavibamycins was restored. This excludes any kind of "upstream" or "downstream" effect.

Example 7 Analysis of the newly formed avilamycin derivatives by S. viridochromogenes GW4 and S. viridochromogenes GW4-AM1

Avilamycin A (M + Na: 1425) and avilamycin C (M + Na: 1427) were detected in extracts from S. viridochromogenes Tü57 by liquid chromatography (LC) mass spectrometry analysis. Avilamycin C was the main component. Measurement at high resolution showed that both compounds contain 2 chlorine atoms, which can be recognized by their typical isotope pattern. The mass of the two main compounds formed by S. viridochromogenes GW4 was 1411 (M + Na) and 1413 (M + Na) (Fig. 111), which shows that aviG4 really codes for a methyl transferase. The major products of the GW4 mutant were isolated, ethylated by treatment with ethyl iodide and hydrolyzed using methanol and hydrochloric acid. The reaction products were analyzed by GC-MS. The mass spectrum of this sample showed several peaks (Fig. 112). The peak by m / z 436 corresponds to the D-olivosyl ester of dichloro-di-O-ethyl-orsellinic acid and most of the other peaks (m / z 405, m / z 275, m / z 247) corresponded to fragments derived from orsellinic acid. Run out the rest (Fig. 112). This suggests that the difference between avilamycin A (C) and the new derivative, gavibamycin A1 (A3), results from a change in the structure of the orsellic acid residue.

Gavibamycin A1 and A3 correspond to the general formula I with the following meaning for the radicals R1-R9:

S. viridochromogenic GW4-AM1 was also analyzed by HPLC-MS. The mass of the two major avilamycin derivatives was 1343 (M + Na) and 1345 (M + Na). When comparing the isotope pattern of the main derivatives from mutant GW4, the isotope pattern of the main products of mutant GW4-AM1 showed no specific signals for chloride ions (Fig. 111), which indicates that the inactivation of aviH leads to the loss of both chloride atoms , The new derivatives were named Gavibamycin B 1 (avilamycin A analog) and Gavibamycin B3 (avilamycin C analog). Gavibamycin B1 and B3 correspond to the general formula I with the following meaning for the radicals R1-R9:

Example 8

Biological properties of Gavibamycin A3

The antimicrobial spectrum of Gavibamycin A3 was determined and compared with that of Avilamycin A. The "broth microdilution" method was used in accordance with the regulations of the national committee for clinical laboratory standards. Both metabolites showed antibiotic activity against Bacillus subtilis, Staphylococcus aureus ATCC6538, Staphylococcus aureus ATCC6538P, Staphylococcus aureus ATCC29213.2, Staphylococcus aureus. 1, Enterococcus faecalis ATCC29212, Enterococcus faecalis H-7-6 and Streptococcus pneumoniae ATCC49619.

Initial tests further show that Gavibamycin A3 is somewhat more active against various Staphylococcus aureus strains than Avilamycin A and it also appears to be much more hydrophilic than can be seen from the Rf values. The non-chlorinated gavibamycin derivatives were also antibiotic active.

Example 9

The mutants S. viridochromogenes GW2 and GW5 and the avilamycin derivatives according to the invention formed by them As with the variants GW4 and GW4-AM1, GW2 and GW5 were produced based on S. viridochromogenic mutants, R3 = OH being in each case of all avilamycin derivatives synthesized by both mutants, and mutants GW2 R6 = H and in the products the mutant GW5 R9 = H. The procedure was completely analogous to that in Examples 4 to 6, in particular 5, so that in the case of GW2, a double mutant, in addition to aviG4 (see Example 4), aviG2 was also genetically modified (switched off). In the mutant GW5, also a double mutant, aviG4 was also genetically modified (switched off) in addition to aviG4. From the products according to the invention of these mutants GW2 and GW5 it can be seen that the corresponding methyltransferases (aviG2 or aviGδ and in each case aviG4) have been switched off.

Example 10

Parenteral application form

5 g of Gavibamycin A3 are dissolved in 1 liter of water, if necessary using a pharmaceutically acceptable solubility improver, for injections at room temperature and then adjusted to isotonic conditions by adding anhydrous glucose for injections.

For an average patient of approx. 65 kg body weight, 0.5 ml, for example, 2.5 mg or 40 μg / kg are applied. The dose administered showed no contraindication and proved to be well tolerated by the patients. A dose of Gavibamycin A3 up to 20 times higher was also found to be toxicologically safe and well tolerated. Example 11

In summary, it can be stated that, according to the invention, a detailed sequence analysis of the avi gene set has several features which suggest a model of a biosynthetic pathway to complex oligosaccharide antibiotics according to the invention. The function of the genes responsible for sugar biosynthesis can be derived from their amino acid sequences, which are similar to those proteins which are involved in the biosynthesis of D-olivose in other organisms. As described for the biosynthesis of D-olivose in Streptomyces violaceoruber Tü22 (granaticin producer) and Streptomyces fradiae (urdamycin producer), the biosynthesis of glucose-1-phosphate begins, which leads to dTDP-D-olivose and dTDP-2-deoxy -D- Evalose is converted by several enzymes. A new feature in this pathway is that three different dNDP-hexose-4,6-dehydratase genes are involved. Based on sequence homologies, AviEl is a dTDP-glucose-4,6-dehydratase and AviE3 is a GDP-mannose-4,6-dehydratase, indicating that the biosynthesis of some of these different sugar units starts from different nucleotide-bound hexose pools. Based on the structure of avilamycin A and also indicated by the supposed function of some gene products, the biosynthesis of D-lyxose even starts from a third sugar pool, so that it could be a product of the pentose-phosphate metabolic pathway. Residue H of avilamycin A was originally described as methylurekanate derived from 2,3-di-O-methylene-4, δ-dihydroxyhexanoic acid. However, the sequence analysis according to the invention shows that methyl urecanate is also the product of a biosynthetic sugar metabolism pathway. All of this, based on the number of sugar units, suggests that the avilamycin cluster is six Has glycosyltransferase genes. However, only four have been found in the avilamycin cluster according to the invention. A possible explanation could be the involvement of one or more glycosyltransferases in several synthetic steps or the involvement of glycosyltransferases that are encoded in regions outside of this gene cluster. Three out of four glycosyltransferases are more reminiscent of glycosyltransferases for the biosynthesis of O-antigen structures or cell wall polysaccharides, which can be explained by the polysaccharide-like structure of avilamycins.

The avi metabolic pathway also contains other interesting features: two orthoester bridges and one methylene bridge. Taking into account the oxidative nature of these COC arrangements, the - ketoglutarate-dependent oxygases AviO1, AviO2 and AviO3 should catalyze the formation of this rare bond. It is therefore described according to the invention that such enzymes use molecular oxygen as a direct electron acceptor for the oxidation through the use of α-ketoglutarate as cosubstrate and thereby ultimately produce the CO-C bonds, succinate and CO ₂ . Avilamycin A heptasaccharide is modified by methylation, by coupling of acetate, by coupling of dichloroisoevernic acid and by coupling from an isobutyrylic unit. Six methyltransferase genes are present in the cluster, which is sufficient in number for avilamycin biosynthesis, while the genes responsible for the coupling of the other residues have not yet been localized. Interestingly, it was found according to the invention that aviB1 and aviB2 encode enzymes which are similar to the alpha and beta chains of component 1 of the 2-oxo acid dehydrogenase complexes. These complexes are usually composed of 3 enzymatic units, namely the TPP-dependent dyhydrogenases (heterotetramers (0 ^ 2), Dihydrolipoamide acetyltransferases (homomultimers) and

Dihydrolipoamide dehydrogenases (homodimers). The ORFs coding for the latter components of these complexes have either not yet been localized within the cluster, are not in the cluster or are not used at all for the biosynthesis of avilamycins.

Gavibamycin A3 was also tested for its antibiotic activity. The first MIC experiments showed that Gavibamycin A3 is somewhat more active than Avilamycin A against various Staphylococcus aureus strains. In addition, it is somewhat more hydrophilic than avilamycin A, as demonstrated by the retention factors from the DC and HPLC analysis. The non-chlorinated gavibamycin derivatives are also antibiotic active.

Literature:

- Buzzetti, F., Eisenberg, F., Grant, HN, Keller-Schierlein, W., Voser, W. _f Zahnner, H. (1968) Experientia 24 (4): 320-323. - Foster, DR, Rybak, MJ (1999) Pharmacotherapy 19: 1111-1117

- Langer, E. (1987) Comparative studies on the mode of action of avilamycin A and eveminomicin B. Diploma thesis from the Faculty of Biology, Eberhard-Karls-University Tübingen.

- Mertz, J.L., Peloso, J.S., Barker, B.J., Babbitt, G.E., Occolowitz, J.L., Simson, V.L., Kline, R.M. (1986) Antibiot 39 (7): 877-887.

- Walker, CA. (1976) Eveminomycin B - a possible site of action. I6th Interscience Conference on antimicrobial agents and chemotherapy, Abstract 116

- Weitnauer, G., Bechthold, A. (1999) PZ Prisma 2: 117-126. - Wolf, H. (1973) FEBS Lett 36 (2): 181-186.

- Wright, D. (1979) Tetrahedron Lett. 36: 1207-1237

- Zahnner, H. (1999) Tübingen, personal communication

Claims

claims

Avilamycin derivative according to general formula I, also in the form of its diastereomers or enantiomers or racemic or other mixtures or pure diastereomers and / or enantiomers,

, where independently with the following exception

R1 is selected from H, COCHs, COC H ₉ , COCH (CH ₃ ) ₂ or COCH ₂ CH ₃ ,

R2 is selected from H, CHO, COCH ₃ or CH (OH) CH ₃ ,

R3 corresponds to OCH ₃ ,

R4 corresponds to Cl,

R5 corresponds to Cl,

R6 corresponds to CH ₃ ,

R7 corresponds to H, CH ₃ or CH ₂ OH, R8 CH ₃ corresponds

and

R9 corresponds to CH ₃ ,

R3 is to be replaced by OH,

R4 is to be replaced by H,

Rδ is to be replaced by H,

R6 is to be replaced by H,

R8 is to be replaced by H.

and or

R9 is to be replaced by H,

with the proviso that R1-R9 cannot simultaneously assume the meanings according to the respective combination in one of the compounds 1-4:

Avilamycin derivative according to claim 1, characterized in that at least R3 is to be replaced by OH, with the proviso that R1-R9 cannot simultaneously take on the meanings according to the combination in compound 1:

Avilamycin derivative according to one of claims 1 or 2, characterized in that at least R4 and Rδ in formula I are to be replaced by H.

Avilamycin derivative according to one of Claims 1 to 3, characterized in that at least R6, R8 and / or R9 is / are to be replaced by H, with the proviso that R1-R9 do not simultaneously have the meanings according to the combination in compound 3 or cannot simultaneously take on the meanings according to the combination in compound 4:

Avilamycin derivative according to one of Claims 1 to 4, characterized in that on the one hand at least R3 is to be replaced by OH and on the other hand at least R4 and Rδ are to be replaced by H or at least R6, R8 and / or R9 is to be replaced by H /are. Avilamycin derivative according to general formula I, also in the form of its diastereomers or enantiomers or racemic or other mixtures or pure diastereomers and / or enantiomers, selected from compounds in which R1-R9 are each combined as follows:

LS

SI860 / I0d3 / X3d 9Ct890 / z0 OΛV

, preferably

7. Avilamycin derivative, obtainable in that, in a cultivable cell, the genes or enzymes necessary for the synthesis of an orthosomycin base body consisting of (a) a terminal dichloroisoevernic acid residue (A in formula I) and

has, at least one nucleic acid, the sequence of which corresponds to at least 9δ%, preferably 97%, in particular exactly, the nucleic acid sequence according to one of Figures 1 to 64, is genetically modified, deleted or not expressed, the modified cell is cultivated, the culture supernatant is obtained and is worked up, the avilamycin derivative (s) is purified and isolated and, if appropriate, various derivatives are separated,

8. Avilamycin derivative according to claim 7, characterized in that the cultivable cell is selected from a cell of the Streptomyces viridochromogenes type or a cell which, with the exception of the genetically modified, deleted or not expressed nucleic acid / s, the nucleic acids according to a sequence of one of the consecutive No. 1-δ4 (according to Table 1 in connection with Fig. 1) or to this at least 9δ%, preferably 97%, contains homologous nucleic acids or contains the gene cluster according to Fig. 109, preferably selected from a cell of the type Streptomyces viridochromogenes, in particular a cell of the type Streptomyces viridochromogenes Tu 67.

Avilamycin derivative according to one of claims 7 or 8, characterized in that the changed nucleic acid (s) for a methyltransferase and / or for a halogenase encoded / n.

10. Avilamycin derivative according to claim 9, characterized in that the sequence (s) of the changed nucleic acid (s) prior to the change is at least 96%, preferably 97%, in particular exactly, the nucleic acid sequence (s) of at least one of the sequences with ongoing No. 1 or 2-7 (according to Table 1 in connection with Fig. 1), preferably one of the sequences with consecutive No. 1, 2, 4 or 6 (Table 1 in connection with Fig. 1) corresponds in particular to the sequence with sequential No. 2 or the sequences with sequential No. 2 and No. 1, No. 2 and No. 4 or No. 2 and No. 6 (according to Table 1 in connection with Fig. 1).

11. Avilamycin derivative according to one of claims 7 to 10, characterized in that the change in the nucleic acid / s leads to the fact that the or by the genetically modified / n encoded / n protein / s or polypeptide / s after the genetic modification is no longer synthesized.

12. Avilamycin derivative, according to one of claims 1 to 5, 6 or 7 to 11, characterized in that it is more hydrophilic than avilamycin A or C or evernimycin (ziracin).

13. Nucleic acid in its sequence to at least 9δ%, preferably 97%, in particular exactly, according to the nucleic acid sequence according to one of the sequences with the running numbers 1 to 51 (according to Table 1 in conjunction with Fig. 1).

14. Nucleic acid according to claim 13, characterized in that the nucleic acid to at least 95%, preferably 97%, in particular exactly, the nucleic acid sequence according to one of the sequences with sequence numbers 1 to 32 and 48 to 61 (according to table

1 in conjunction with Fig. 1), preferably 1 to 7, in particular 1, 2, 4 or 6, corresponds.

15. Gene cluster containing "open reading frames", preferably 54, which have at least 95%, preferably in their nucleic acid sequence

97%, in particular exactly, according to the nucleic acid sequences correspond to the sequences with sequence numbers 1 to 64 (according to Table 1 in connection with Fig. 1) and which are arranged on a nucleic acid strand, preferably according to Fig. 109.

16. Protein or polypeptide in its amino acid sequence to at least 95%, preferably 97%, in particular exactly, in accordance with the amino acid sequence according to one of the sequences with serial numbers 55-104 (according to Table 1 in conjunction with Fig. 1).

17. Protein or polypeptide according to claim 16, characterized in that the protein or polypeptide is at least 95%, preferably 97%, in particular exactly, the nucleic acid sequence according to one of the sequences with sequence numbers 56 to 86 (according to Table 1 in conjunction with Fig. 1 ), preferably 56 to

61, in particular 55, 56, 58 or 60, corresponds.

18. Protein or polypeptide encoded by a nucleic acid according to one of claims 13 or 14.

19. Genetically modified cell containing at least one non-endogenous nucleic acid according to one of claims 13 or 14, a non-endogenous gene cluster according to claim 15 and / or a non-endogenous protein or polypeptide according to one of claims 16-18.

20. Cell containing at least one genetically modified nucleic acid, the sequence of which before the change is at least 95%, preferably 97%, in particular exactly, of the nucleic acid sequence according to one of the sequences with current

Nos. 1 to 54 (according to Table 1 in conjunction with Fig. 1) corresponded.

21. Cell of the Streptomyces viridochromogenes type, preferably of the Tü57 subtype, characterized in that at least one of the nucleic acids with a sequence according to one of the sequences with sequence numbers 1-54 (according to Table 1 in conjunction with Fig. 1) has been genetically modified or deleted.

22. Cell according to claim 21, characterized in that at least one of the nucleic acids with a sequence according to one of the sequences with sequence number 1 or 2-7 (according to Table 1 in conjunction with Fig. 1), preferably with one of the sequences with sequence number. 1, 2, 4 or 6 (Table 1 in connection with Fig. 1), in particular with a sequence with consecutive No. 2 or with the sequences with consecutive No. 2 and No. 1, No. 2 and No. 4 or No. 2 and No. 6 (according to Table 1 in connection with Fig. 1), has been genetically modified or deleted.

23. Cell according to claim 21 of the mutant type Streptomyces viridochromogenes GW4, Streptomyces viridochromogenes, GW4-AM1, Streptomyces viridochromogenes GW2 or Streptomyces viridochromogenes GW5, characterized in that of the mutant type Streptomyces viridochromogenes GW4 Avilamycin derivatives are synthesized in which derivatives R3 OH is of the mutant type Streptomyces viridochromogenes GW4-AM1 avilamycin derivatives are synthesized in which R3 = OH, R4 = H and R5 =

H are, the mutant-type Streptomyces viridochromogenic GW2 avilamycin derivatives are synthesized in which R3 = OH and R6 = H and the mutant-type Streptomyces viridochromogenic GW5 avilamycin derivatives are synthesized in which R3 = OH and R9 = H ,

24. Use of a nucleic acid according to one of claims 13 or 14, a gene cluster according to claim 15, a protein or polypeptide according to one of claims 16 to 18 and / or a cell according to one of claims 19 to 22 for the production of an avilamycin derivative, preferably according to one of claims 1 to 12.

25. A process for the preparation of avilamycin derivatives according to one of claims 1 to 6, characterized by the following steps:

(a) a terminal dichloroisoevernic acid residue (A in formula I) and

has at least one nucleic acid, the sequence of which is at least 95%, preferably 97%, in particular exactly, the nucleic acid sequence according to one of the sequences with sequence numbers 1 to 54 (according to Table 1 in conjunction with FIG. 1) corresponds, genetically modified, deleted or not expressed,

(4) the culture supernatant is worked up and the avilamycin derivative (s) formed is purified and isolated, (6) if appropriate, different derivatives are separated.

26. The method according to claim 25, characterized in that the cultivable cell is selected from a cell of the type Streptomyces viridochromogenes or a cell which, with the exception of the genetically modified, deleted or unexpressed nucleic acid, the nucleic acids according to a sequence with consecutive No. 1- 54 (according to Table 1 in conjunction with Fig. 1) or at least 95%, preferably 97%, of homologous nucleic acids or contains the gene cluster according to claim 14, is preferably selected from a cell of the type Streptomyces viridochromogenes, in particular a cell of the type Streptomyces viridochromogenic Tu 57.

27. The method according to any one of claims 25 or 26, characterized in that the changed nucleic acid / s codes for a methyl transferase and / or for a halogenase / s.

28. The method according to claim 27, characterized in that the sequence (s) of the changed nucleic acid (s) before the change to at least 95%, preferably 97%, in particular exactly, the nucleic acid sequence (s) of at least one of the sequences with consecutive no 1 or 2-7 (according to Table 1 in connection with Fig. 1), preferably one of the sequences with consecutive numbers 1, 2, 4 or 6 (Table 1 in connection with Fig. 1) corresponds in particular to the sequence with sequential No. 2 or the sequences with sequential No. 2 and No. 1, No. 2 and No. 4 or No. 2 and No. 6 (according to Table 1 in conjunction with Fig. 1).

29. The method according to any one of claims 26 to 28, characterized in that the change in the nucleic acid / s leads to the fact that the or by the genetically modified nucleic acid / s encoded / n protein / s or polypeptide / s according to the genetic engineering Change is no longer synthesized.

30. Medicament containing avilamycin derivatives according to one of claims 1 to 12 and optionally suitable additives and / or auxiliaries.

31. Use of an avilamycin derivative according to any one of claims 1 to 12 for the manufacture of a medicament with an antibiotic effect for the treatment or for the manufacture of a medicament for the treatment of diseases, for example.

Infectious diseases.